A dual-purpose polymerase engineered for direct sequencing of pseudouridine and queuosine

More than 170 posttranscriptional RNA modifications are so far known on both coding and noncoding RNA species. Within this group, pseudouridine (psi) and queuosine (Q) represent conserved RNA modifications with fundamental functional roles in regulating translation. Current detection methods of these modifications, which both are reverse transcription (RT)-silent, are mostly based on the chemical treatment of RNA prior to analysis. To overcome the drawbacks associated with indirect detection strategies, we have engineered an RT-active DNA polymerase variant called RT-KTq I614Y that produces error RT signatures specific for psi or Q without prior chemical treatment of the RNA samples. Combining this polymerase with next-generation sequencing techniques allows the direct identification of psi and Q sites of untreated RNA samples using a single enzymatic tool.